It is known to enhance the effectivity of immunizations by administering the immunogen repeatedly. The second and, if necessary, subsequent booster injections induce high titres of protecting antibodies.
It is also known to enhance the effectivity of an immunogen by a pretreatment of the host with a subunit of the native immunogen. This so-called priming effect has been shown, for example, with a peptide comprising part of the amino acid sequence of the B subunit of cholera toxin. This peptide was coupled to a protein carrier, and the conjugate was used for priming immunizations. After priming, injection with a sub-immunizing amount of cholera toxin led to a substantial level of neutralizing antibodies. [Jacob et al., The EMBO Journal 4 (1985), pages 3339-3343 ].
In case of polio vaccine, Van Wezel et al. [Dev. Biol. Stand. 55 (1984), pages 209-215] have shown a similar priming effect. Injection of poliovirus capsid proteins isolated by means of SDS-PAGE, followed by an injection with inactivated polio vaccine resulted in stimulation of the formation of neutralizing antibodies.
In 1983 Emini et al. [Nature 304 (1983), pages 699-703] have described the priming principle in connection with peptides consisting of 5-10 amino acids coupled to carrier proteins. The sequence of the peptides corresponded with parts of the capsid protein I of type 1 poliovirus (strain: Mahoney). In all cases a stimulation of the neutralizing response to type 1 poliovirus was observed.
Viruses of the picornavirus family are RNA containing viruses. Their diameter is about 30 nm and their protein capsid has the form of an icosaeder.
Based on physico-chemical characteristics the family is divided into four genera:
a. enterovirus (e.g. polio, hepatitis A, echo and coxsackievirus), PA0 b. cardiovirus (e.g. encephalomyocarditis and Mengovirus), PA0 c. rhinovirus and PA0 d. aphtovirus (e.g. foot-and-mouth disease virus).
Within the members of the genera a differentiation into types is made. For example, there are three types (1 to 3) of poliovirus, at least 89 types of rhinovirus and 7 types of foot-and-mouth-diseasevirus.
The poliovirion contains 60 protomers. Each of these protomers consists of four capsid proteins (VP1 to VP4). Capsid proteins VP1, VP2 and VP3 are positioned at the surface of the virion, whereas VP4 is located in the interior of the protein capsid. The position of the three surface positioned capsid proteins is disclosed in Hogle et al. Science 229 (1985), pages 1358-65. The three-dimensional structure of these three capsid proteins is essentially identical. In their native conformation the capsid proteins contain four loops and eight so-called beta-sheets.
The amino acid sequence of the capsid proteins of a number of picornaviruses is known. Picornaviruses having a known primary structure are the three types of poliovirus [Nature 291 (1981), pages 547-553; J. Mol. Biol. 174 (1984), pages 561-585; Proc. Natl. Acad. Sci. USA 81 (1984), pages 1539-1543], hepatitis A (Proc. Natl. Acad. Sci. USA 82 (1985), pages 2627-2631], two types of rhinovirus [Nucl. Ac. Res. 13 (1985) pages 2111-2126; Proc. Natl. Acad. Sci. USA 82 (1985), pages 732-736], coxsackievirus B3 [Vir. Res. 3 (1985), pages 263-270], encephalomyocarditisvirus [Nucl. Ac. Res. 12, (1984) pages 2969-2985], and a number of foot-and-mouth disease virus strains [Gene 17 (1982), pages 153-161; Nucl. Ac. Res. 12 (1984), pages 6587-6601]. Comparison of these sequences shows that the primary structure of the three types of poliovirus is more strongly related to that of the rhinoviruses than to that of hepatitis A. Hepatitis A belongs, just as poliovirus, to the genus of enteroviruses. This shows that the relationship within the genus is not necessarily stronger than that outside the genus. Emini et al. (Virology 140 (1985), pages 13-20) have shown by means of immunochemical techniques that there are relationships between the capsid proteins of a number of enteroviruses and those of rhinovirus 2. Capsid protein 3 showed the strongest cross-reactivity
Immunization with inactivated polio vaccine (generally formalin inactivated virus suspension of the three types of poliovirus) results in the formation of neutralizing antibodies. These antibodies bind with the intact virus. The position of the antigenic determinants giving rise to the formation of neutralizing antibodies has been determined by means of the so-called "escape mutans" (Nature 301 (1983), pages 674-679; J. Virol. 57 (1986), pages 246-257). It appears in practically all cases that these determinants occur in the loops of the capsid proteins. The capsid proteins of the poliovirus show a strong mutual interaction. As a consequence of this interaction denaturing conditions have to be used for the isolation of the capsid proteins. Injections with the purified capsid proteins of poliovirus does not result in the formation of significant titres of neutralizing antibodies. This lack of capability to induce the formation of neutralizing antibodies has to be ascribed to the fact that the threedimensional structure of the isolated capsid proteins strongly differs from the threedimensional structure of the proteins in the virus.
Toyoda et al. in J. Mol. Biol. 1984, pages 561-585 disclose the primary structure of the capsid proteins of the three types of poliovirus (of each type the Sabin strain was analyzed). Comparison of these structures shows strong sequential homologies for the three types. The positions in the capsid proteins where differences are observed generally are positioned in the loops of the capsid proteins.
At least two lymphoid cell populations are involved in the formation of antibodies after injection of immunogens: The B cell and the helper T cell. The B cell recognizes the antigenic determinant and the helper cell recognizes the carrier determinant after processing of the immunogen. Eventually, the B cell, after differentiation into a plasma cell, is responsible for the formation of antibodies directed to the antigenic determinant. Based on these immunological data, Gupta et al. (Proc. Natl. Acad. Sci. USA 83 (1986), pages 2604-2608) have suggested that the B cell recognizes the antigenic determinant against which neutralizing antibodies are formed, and that the helper T cell recognizes the rest of the virus. As stated above, the rest of the virus contains a large number of common sequences. The recognition by the helper T cell is considerably less conformation-dependent than the recognition by the B cell. Based on this data and the occurrence of common sequences in native immunogens it seems to be probable that T helper cells recognizing these common structures will be formed after immunization with native proteins or after injection with proteins or peptides showing sequential homology with said native proteins.
The stimulation is observed not only with the virus type homologous with the capsid protein (homotypical), but also for the other virus types (heterotypical). The degree of priming stimulation practically corresponds with that of the booster effect when the vaccine is injected twice. The denatured capsid proteins are not or hardly capable of inducing the formation of neutralizing antibodies. It is believed that this priming stimulation obtained by injection with the carrier portion of an immunogen followed by an injection with the complete immunogen results in stimulation of the antibody response to the hapten (antigenic determinant). Most probably, the common primary structures are responsible for the heterotypical stimulation by the capsid proteins.